[Journal Club] Elucidation of the mechanism of [2Fe-2S] clusters biosynthesis
Iron-sulfur (Fe-S) clusters are ubiquitous metallocofactors constituting the active site of a multitude of enzymes and proteins involved in electron transfer, catalysis, sulfur donation and signalling. They are made of iron and sulfide ions assembled into diverse structures. The [2Fe-2S] and [4Fe-4S] clusters are the most common forms in organisms. They are synthesized by multi-protein machineries which have remained highly conserved during evolution. The iron-sulfur cluster (ISC) assembly machinery present in eukaryotes and prokaryotes synthesizes [2Fe-2S] clusters, which serve as building blocks for the assembly of [4Fe-4S] clusters. The core ISC machinery assembles [2Fe-2S] clusters on the scaffold protein IscU, which requires iron provided by an unknown source, sulfur provided in the form of cysteine bound persulfides (Cys-SSH) by the cysteine desulfurase IscS, and electrons provided by the ferredoxin–ferredoxin reductase complex Fdx-FdxR from NADPH. Then, specialized chaperones transfer [2Fe-2S] clusters to recipient acceptors. Despite previous studies on the core assembly machinery, the mechanistic details of the [2Fe-2S] cluster assembly process have remained poorly understood due to the experimental difficulties in trapping the relevant intermediates.
The team Biochemistry of Metalloproteins and Associated Diseases headed by Benoit D’Autréaux at the Institute of Integrative Biology of the Cell in Gif-Sur-Yvette (I2BC, UMR 9198, CNRS – CEA – Paris-Saclay University) managed to dissect this process step by step and to isolate several key intermediates using a functional reconstitution of the Escherichia coli ISC machinery. They used a combination of biochemical techniques to trap these intermediates: anaerobic reconstitution, persulfide detection assays, kinetics, UV-visible, circular dichroism, and to characterize them by spectroscopic methods: electron paramagnetic spectroscopy (EPR), nuclear magnetic resonance (NMR) and native mass spectrometry (nMS) in collaboration with teams at Aix-Marseille University (Bénédicte Burlat, BIP), Gif-Sur-Yvette (Christina Sizun, ICSN) and Strasbourg (Sarah Cianférani IPHC) and. They show that the assembly of [2Fe-2S] clusters is initiated by iron binding to IscU, which triggers persulfide insertion by IscS in the vicinity of the iron-binding site of IscU upon the formation of a complex between IscU and IscS. The persulfide in IscU binds to the iron center and is cleaved into sulfide by the Fdx-FdxR complex, which leads to the formation of a Fe-SH intermediate, referred to as the [1Fe-1S] precursor. Then, IscU dissociates from IscS, dimerizes and generates a bridging [2Fe-2S] cluster by fusion of two [1Fe-1S] precursors. The IscU dimer ultimately dissociates into a monomer, ready to transfer its [2Fe-2S] cluster to acceptors. The data also indicate that the bridging cluster is initially in the super-reduced state [2Fe-2S]0 and releases two electrons to the ferredoxin enzyme, thereby leading to an oxidised [2Fe-2S]2+ state as the final product.
These data provide a comprehensive description of mechanism of [2Fe-2S] clusters assembly by the bacterial ISC machinery, highlighting the formation of key intermediates through a tightly concerted process. This stepwise dissection further supports findings in eukaryotes, including iron loading, persulfidation and dimerization of IscU, which point to an evolutionary conservation of the assembly process.
Article: https://www.nature.com/articles/s41589-024-01818-8
News and Views: https://www.nature.com/articles/s41589-024-01835-7